DNA topoisomerases am ubiquitous enzymes which catalyze the breakage and rejoining of the DNA phosphodiester backbone (J. C. Wang, Annu. Rev. Biochem., 54, pages 665-697, 1985; P.-H. Vosberg, Curr. Top. Microbiol. Immunol, 114, pages 19-102, 1985). These reactions, together with an intervening strand passage event, allow this class of enzymes to alter DNA topology. Intermediates in the strand passage reaction may involve either single or double stranded breaks allowing the classification of topoisomerases into type I and type II enzymes respectively. In eukaryotic cells, including human cells, a single type I enzyme has been described (J. C. Wang, Biochem. Biophys. Acta., 909, pages 1-9, 1987). The bacteria E. coli is known to possess two type I enzymes. One, encoded by the topA gene, is responsible for the major DNA relaxing activity of the cell and is essential in an otherwise wild type E. coli (G. J. Pruss et al., Cell, 31, pages 35-42, 1982; S. DiNardo et al., Cell, 32, pages 43-51, 1982). The second E. coli enzyme, topoisomerase III, is a minor activity encoded by the topB gene which appear to be completely dispensable (H. Hiasa et al., J. Biol. Chem., 269, pages 2093-2099, 1994).
Although the E. coli and human topoisomerase I both catalyze the relaxation of negatively supercoiled DNA, they differ in the details of the reactions they catalyze and share no amino acid sequence homology. The human topoisomerase I ("hTOPI") shows a preference for binding double stranded DNA and proceeds by making a single covalent 3'-phosphodiester intermediate to a tyrosine of the enzyme (M.D. Been et al., J. Mol. Biol. 180, pages 515-531, 1984; W. Baase et al., Biochemistry, 13, pages 4299-4303, 1974; J. J. Champoux, Proc. Natl. Acad. Sci. U.S.A., 74, pages 3800-3804, 1977). The E. coli enzyme ("eTOPI") demonstrates a preference for binding at the junction of double and single stranded regions and proceeds using a single 5'-phosphodiester intermediate (J. C. Wang, J. Mol. Biol., 55, pages 532-533, 1971; K. Kirkegaard et al., J. Mol. Biol., 185, pages 625-637, 1985; R. E. Depew et al., J. Biol. Chem., 253, pages 511-518, 1978). The eTOPI enzyme is very efficient at relaxing highly negatively supercoiled DNA and shows progressively decreasing activity as the substrate DNA becomes more relaxed (J. C. Wang, J. Mol. Biol., 55, pages 531-533, 1971; K. Kirkegaard et al., Proc. Natl. Acad. Sci. U.S.A., 184, pages 625-637, 1985). The hTOPI enzyme relaxes both negatively and positively supercoiled DNA to completion (W. Baase et al., Biochemistry, 13, pages 4299-4303, 1974). The hTOPI enzyme is the target of the antitumor drug camptothecin (CMPT) which traps the covalent phosphotyrosine intermediate of the strand passage reaction (Y.-H. Hsiang, et al., J. Biol. Chem., 260, pages 14873-14878, 1985.). E. coli eTOPI enzyme (K. Drlica et al., Biochemistry, 27, pages 2253-2259) is resistant to CMPT.
It previously has been shown that plasmid born copies of both the yeast (M.-A. Bjornsti et al., Proc. Natl. Acad. Sci. U.S.A., 84, pages 8971-8975, 1987) and human topoisomerase I (M.-A. Bjornsti et al., Cancer Research, 49, pages 6318-6323, 1989) coding sequences possess the capacity to complement a conditional defect in the bacterial topA gene. Subsequent to this study the same group of researchers have pursued the development of yeast model systems for the study of the eukaryotic topoisomerase I (J. Nitiss et al., Proc. Natl. Acad. Sci. U.S.A., 85, pages 7501-7505, 1988; M.-A. Bjornsti et al., Cancer Research, 49, pages 6318-6323, 1989). Heretofore, no E. coli system useful as a model for the expression of hTOPI has been reported. This present invention concerns an E. coli system for the controlled functional expression of hTOPI.